First Report of Phytophthora Taxon Walnut in Lombardy, North Italy.

The park Boscoincittà, Milan, North Italy (136 m a.s.l., 45°29'06″ N, 9°5'32″ E), has an area of 110 ha and includes tree stands, wood clearings, trails, and watercourses. Recently, common walnut (Jugland regia) trees in the park have begun to suffer from a progressive dieback that has caused roughly 90% mortality. Aerial symptoms were stunted growth, loss of vigor, crown thinning, and bark cankers with tarry exudates on the lower stem. The xylem tissue of trees showed large necrosis and flame-shaped discoloration below the bark. Since the dieback seemed caused by Phytophthora, samples were taken from three symptomatic trees and, by baiting, from the nearby soil and watercourses. Isolations from apple baits were carried out after a week. Isolations taken from tissue at the edge of active lesions of the trees were transferred on the selective medium V8A-PARPNH (1) and incubated at 24°C. Cottony colonies appeared after 3 days and single hyphal tip derivatives were transferred to V8A for a further 4 to 7 days. Fragments (1 cm2) of mycelium of the subcultured colonies were then placed in filtered (Ø 0.22 µm nitrocellulose filters, Millipore) pond water. Three isolates were retrieved within 24 h, two from tree tissue and one from water. These produced ovoid, non-papillate sporangia, of which 30 per isolate were measured. Sporangia averaged 52.5 ± 9.6 × 32.9 ± 4.7 µm (range 30.8 to 67 × 22.5 to 42.8) with a l:b ratio of 1.59 ± 0.19 (range 1.27 to 2.05), and exit pores of 11.1 ± 1.7 µm (range 7.31 to 14.21). External proliferation from previously emptied sporangia and hyphal swellings were observed on V8A. On V8A, colonies had optimal growth at 32°C (5.7 ± 0.8 mm d-1) with a maximum at 37°C. Colonies had a chrysanthemum-shaped, scanty fluffy aspect on PDA. Isolates were identified as Phytophthora taxon walnut on the basis of macro- and micro-morphology and sequence information from the ITS-rDNA region, that was amplified with primers ITS6 and ITS4 (3) after DNA extraction with a commercial kit (Sigma Aldrich). A region of the cox1 gene of isolate B164 was also amplified with primers OomCoxILevup and Fm85mod (4) and sequenced (GenBank Accession No. KC291584), but this was irrelevant for identification purposes because that gene region has not been sequenced for other isolates of this taxon. A BLAST search in GenBank and the Phytophthora database revealed a 99% identity of the ITS-rDNA from our isolate B164 (KC291550) with the P. taxon walnut isolate P532 (AF541910) (2). Inoculation trials were conducted on 10 detached leaves. A little lesion was produced with a sterile scalpel on the lower leaf surfaces and a 0.5 cm Ø agar plug was placed over the wounds. Necrotic lesions averaged 3.7 ± 1.6 × 2.0 ± 0.5 cm after 1 week of incubation at 20°C in the dark. Control leaves showed no symptoms. Re-isolations on V8-PARPNH agar confirmed Phytophthora taxon walnut as the causal agent. Members of the Phytophthora genus grouping with the Phytophthora taxon walnut in clade 6 are mainly reported as saprophytes or pathogens from riparian ecosystems and forests (2). To our knowledge, this is the first report of Phytophthora taxon walnut from Italy. Since the oomycete proved in our growth trial to be distinctly thermophilic, we hypothesize that its spread is being favored by the rising temperatures observed during the last decades in the area. References: (1) Y. Balci et al. Plant Dis. 91:705, 2007. (2) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (3) D. E. L. Cooke et al. Fungal Gen. Biol. 30:17, 2000. (4) G. P. Robideau et al. Bioinformatics 19:1572, 2011.

Phytophthora Taxon Pgchlamydo is a Cause of Shoot Blight and Root and Collar Rot of Viburnum tinus in Italy.

The quarantine pathogen Phytophthora ramorum has recently been found on dying Viburnum tinus in the nursery area of Pistoia, central Italy (43°56'0″ N, 11°1'0″ E) (3). As part of a surveillance program aimed at detecting P. ramorum in this area, the Phytophthora taxon Pgchlamydo was consistently found associated with symptomatic V. tinus. The crowns of these plants were wilted, and some plants also showed root and collar rot and underbark necrosis. Water courses adjacent to the nursery with the infected V. tinus were tested for the pathogen. Samples from seven symptomatic plants were placed on a selective V8A-PARPNH medium within 24 h from sampling. Tissue pieces (2 mm2) of 12 baits (apple fruits) exposed for a week in water bodies were plated on the same medium. Cottony colonies arose after 2 to 3 days of incubation at 23°C in the dark and were transferred to potato dextrose agar (PDA) in purity. Mycelial DNA was extracted with a commercial kit (Sigma-Aldrich). The rDNA ITS region and a portion of the mtDNA cox1 gene were PCR-amplified and the amplicons digested with the restriction enzymes MspI and AluI (for the ITS region) and RsaI (for the cox1 gene region). Isolates R7 from V. tinus, and ES2M5, ES2M11, and ES1M12 from the water bodies belonged to the same taxon based on restriction analysis of both DNA regions coupled with ITS-rDNA sequence homology (GenBank Accession Nos. KJ396773 to 76). A BLAST search in GenBank found that all isolates had a 99% identity in the ITS-rDNA with the Phytophthora ITS Clade 6 member P. taxon Pgchlamydo. Sporangia produced after incubation in filtered pond water for 24 h were mostly ovoid (sometimes obpyriform), non-papillate, non-caducous. Some sporangia were emptied with external proliferation and had hyphal swellings. Thirty sporangia were measured and averaged 42.4 ± 6.2 × 29.9 ± 3.5 µm (range 30.0 to 56.1 × 22.5 to 38.0), with a length/width ratio of 1.4 ± 0.2 (1.2 to 2.0), and exit pores of 11.7 ± 1.5 µm (9.0 to 14.6). Optimum colony growth on V8A at 30°C was 4.4 ± 0.4 mm day-1, and the maximum temperature for growth was 32°C. Inoculation on twigs of Fagus sylvatica and V. tinus produced necrotic lesions of 2.6 ± 0.5 cm (2.1 to 3.5) and 4.7± 0.5 cm (3.8 to 5.6) respectively after 3 weeks of incubation at 23°C in the dark. Inoculation on V. tinus leaves resulted in lesions averaging 3.3 ± 1.1 × 2.1 ± 0.6 cm (range 2 to 5 × 1.5 to 3) after 2 weeks of incubation at 23°C in the dark. Control plant material showed no symptoms.The Phytophthora taxon Pgchlamydo has been reported on several ornamental and woody species, including Arctostaphylos sp., Camellia spp., Laurus nobilis, Buxus sempervirens, Rhododendron sp., Arbutus unedo, Prunus sp., Pseudotsuga sp., and Sequoia sempervirens, in North America and Europe (1,2). This is the first report, to our knowledge, of this taxon on V. tinus in Italy. V. tinus is widely sold in European nurseries, and it is also one of the most common hosts of P. ramorum (4). The fact that V. tinus is a host for both oomycetes, and the two microorganisms induce a similar symptomology (wilt), might complicate the control efforts of the phytosanitary inspection services aimed at restricting P. ramorum foci in Europe. References: (1) C. L. Blomquist et al. Plant. Dis. 96:1691, 2012. (2) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (3) B. Ginetti et al. Plant. Dis. 98:423, 2104. (4) S. Prospero et al. Plant Pathol. 62:1063, 2013.

Foliar Blight and Shoot Dieback Caused by Phytophthora ramorum on Viburnum tinus in the Pistoia Area, Tuscany, Central Italy.

In spring 2013, pot-grown Viburnum tinus plants shipped to an ornamental nursery in Pescia (Pistoia, central Italy, 287 m a.s.l., 43°54'0″ N, 10°41'0″ E) from another local nursery were found to bear disease symptoms. Symptoms included brown to black foliar lesions, later expanding into larger blotches; necrosis of the petioles; shoot wilting and folding; browning of the stems; and necrosis of the cambium. Infected leaves, shoots, and entire plants eventually died. Tissue samples (2 mm2) were cut at the edge of active lesions from tissue of the phloem, the xylem, and the leaves and plated on selective PARPNH V8 agar (V8A) (1). Rose-shaped and finely lobed cottony colonies arose in 2 to 3 days. Mono-hyphal colonies were isolated and transferred to V8A. Square colony pieces (1 cm2) from isolates SB05a and SB05b were placed in filtered pond water after 5 to 7 days. Semipapillate, caducous sporangia with a rounded or conical base were produced within 24 h, individually or in pairs, on each sporangiophore. Sporangia (n = 30 per isolate) were examined: they were 56.2 ± 9.5 × 29.3 ± 4.3 µm (l:b ratio 1.9 ± 0.3). Exit pores averaged 7.0 ± 1.0 µm. Sporangia were ellipsoid (30%), lemon-shaped (28.3%), ovoid (20%), obovoid (16.7%), ampulliform (3.3%), or "peanut-like" (1.7%). Globose chlamydospores, borne intercalarly or terminally, were abundant on both V8A and carrot agar (CA), and were on average 54.7 ± 8.5 µm. Mono-hyphal isolates incubated for 7 days at 23°C were also transferred to CA, corn meal agar (CMA), malt extract agar (MEA), potato dextrose agar (PDA), and V8A. Colonies on these media were identical in shape and appearance to those described in previous reports (2,4). Isolates were identified as Phytophthora ramorum Werres, De Cock & Man in't Veld (4) on the basis of colony type; size, the average l:b ratio and shape of sporangia; and the type and size of the chlamydospores. Isolates were found to be the A1 mating type by pairing them with P. cryptogea BBA 63651 (mating type A2). PCR-amplification of the rDNA ITS region with specific primers Ph1/Ph4 (3) gave fragments of the expected size (GenBank Accession Nos. KF181162 and KF181163). A BLAST search of these ITS sequences in the database found that isolates of P. ramorum were the closest phylogenetically with 100% homology (YQ653034 and HM004221). Pathogenicity tests were conducted on 16 detached V. tinus leaves. A small cut was made aseptically on each of the leaf surfaces and a V8A disc (0.5 cm Ø) with mycelium was placed over the wounds. Control leaves received only sterile V8A discs. Inoculated and control leaves were incubated at 23°C in the dark. Necrotic areas (average 3.5 ± 1.3 cm2) arose on inoculated leaves after 6 days. Control leaves had no symptoms. Re-isolations on PARPNH V8A confirmed P. ramorum as the causal agent. P. ramorum was reported in Italy in 2003 on the exotic Rhododendron yakushimanum (2). This is the first report of the pathogen on a native species (V. tinus) in this country. The Pistoia area is important for nursery gardens and flowers. P. ramorum, which probably arrived on infected plant material, could compromise the export/import trade in stock plants. For this reason, the plant protection services were promptly alerted and the infected plants were destroyed. References: (1) Y. Balci et al. Plant Dis. 91:705, 2007. (2) C. Gullino et al. Inf. Agrar. 19:87, 2003. (3) K. J. Hayden et al. Phytopathology 94:1075, 2004. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.

First Report of Neofusicoccum parvum Associated with Bark Canker and Dieback of Acer pseudoplatanus and Quercus robur in Italy.

Between 2007 and 2011, Acer pseudoplatanus and Quercus robur trees declined in the North Park and the Boscoincittà Park in Milan, Italy (lat. 45° 27' 47″ N, long. 09° 11' 16″ E, elev. 121 m). Symptoms included extensive lengthwise bark cracks, with necrosis of the wood tissue underneath. Isolations were conducted from bark and infected wood after surface sterilization with 10% H2O2 for 10 min, followed by five washings in sterilized water. Tissue was placed on potato dextrose agar (PDA) in petri dishes amended with 0.06 g/l streptomycin at pH 6.5. The dishes were incubated in the dark at 24°C. Colonies reached a diameter of 7 to 8 cm in 7 days. They were whitish grey on the upper surface and blackish at the bottom, and consisted of dense aerial mycelia. The conidia were hyaline, ellipsoid, smooth, non-septate, thin-walled, with their upper end wider than their lower end, and measured 14 to 20 × 4.5 to 7 µm. The causal organism was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (basionym Fusicoccum parvum Pennycook & Samuels; teleomorph Botryosphaeria parva Pennycook & Samuels) based on biometric characteristics, common taxonomic keys, and the sequence information from the rDNA ITS region and the elongation factor 1-alpha (EF1-α) gene (GenBank Accession Nos. HQ893535 and HQ893537, respectively). All morphological traits corresponded to those of the holotype (1), while BLAST searches of the GenBank database revealed for both loci 100% homology with reference sequences. To confirm the involvement of N. parvum in the tree death, and fulfil Koch's postulates, 20 2-year-old seedlings of A. pseudoplatanus and Q. robur (10 each) were artificially infected in May with N. parvum in 2008 and in 2009. A bark portion was removed aseptically and a PDA disc (0.5 cm diameter) of N. parvum mycelium was placed on the wound. Control seedlings (3 per species) received sterile PDA discs. The inoculation site was wrapped in Parafilm for 10 days. After 3 weeks, infected seedlings showed sunken bark lesions associated with necrosis and bark cracks. When the bark was stripped, the wood below these lesions was also necrotic. Control seedlings developed no symptoms. Mycelium of N. parvum was successfully reisolated from the infected tissue. N. parvum is an important member of the Botryosphaeriaceae, a family of cosmopolitan endophytes that have been known for many years (2). The pathogen has been in recent years linked to complex syndromes, in which a prominent symptom is the formation of bark cankers. Although common on fruit crops, it has increasingly been reported on forest trees (3,4). References: (1) P. Crous et al. Stud. Mycol. 55:248, 2006. (2) P. A. Saccardo. Michelia 1:1, 1877. (3) M. E. Sánchez et al. Plant Dis. 87:1515, 2003. (4) B. Slippers and M. J. Wingfield. Fung. Biol. Rev. 21:90, 2007.

First Report of Biscogniauxia mediterranea on English Ash in Italy.

In July 2010, extensive decline of English ash (Fraxinus excelsior L.) was observed near Chiusi della Verna, Tuscany, in central Italy (altitude 956 m a.s.l.; lat. 43° 41' 54″ N, long. 11° 56' 9″E). The symptoms on the tree trunks and leaves included lengthwise bark cracks, detached bark, withering of the crown where the bark was detached, and extensive microphyllia. In September 2010, perithecial stromata were observed in the parts of the tree that had lost their bark. They were applanate and black and fruiting bodies were recognized as Biscogniauxia mediterranea (De Not.) Kuntze (basionym Sphaeria mediterranea (De Not.), Ascomycotina, Xylaraceae), a fungus causing charcoal canker that attacks oaks, Acer spp., Castanea sativa Miller, Fagus sylvatica L., and Platanus acerifolia Willd. The biometric characteristics of 30 stromata were examined. The stromata were slightly convex, ellipsoid, and elongate, 7.2 to 20.5 × 3.5 to 4.2 cm. The perithecia were ovoid to tubular, 0.74 to 0.80 × 0.12 to 0.15 mm; the asci were short and stipitate, 7.9 to 10.0 × 120.2 to 170.4 µm. The ascospores were ovoid, brownish-black, with narrowed and roundish ends, 6.9 to 9.1 × 14.6 to 20.0 µm. Colonies grown on PDA at 25°C for 5 days were grey viewed from the top and black viewed from the dish underside. A comparison with the data in the literature (1) confirmed the macro- and microscopic identification. Traditional identification was further confirmed by sequence information from the rDNA ITS region. A BLAST search of the ITS sequence of our B. mediterranea isolate (GenBank Accession No. JX262798) revealed an exact match (100%) with several reference sequences of the fungus present in the database, mainly from oak hosts. Four branches each of five English ash trees growing in a natural environment were inoculated at the trunk junction with a 10-5 ml ascospore suspension. Control consisted of a branch per tree inoculated with an identical volume of sterile water only. After 25 days, the bark became detached and after a further 15 days the typical black stromata appeared. The pathogen was reisolated from the lesions, confirming Koch's postulates. No symptoms were observed on control branches, which presented healed wounds. B. mediterranea was first detected in 1986 on Quercus cerris L. and Q. pubescens Willd. in the Circeo natural park in central Italy (altitude 50 m a.s.l; lat. 41° 27' 55″44 N, long. 12° 53' 53″52 E). From there it spread north to other parts of the country. All oak species in Italy appear to be susceptible. Its northward expansion is likely associated with the high temperature and water stress that have been affecting the Italian peninsula for the last few years (4). Most recently, B. mediterranea has also been reported on Q. cerris in the Karst region of Slovenia (lat. 45° 43' 03″7 N, long. 13° 45' 20″ 4 E). This confirms its current spread to the more northerly territories, most likely because of ongoing changes in the climate that are creating optimal conditions for its survival in areas that were previously unsuitable to it. References: (1) P. Capretti and L. Mugnai, Inform. Fitopatol. 37:39, 1987. (2) D. Jurc and N. Ogris. Plant Pathol. 55:299, 2006. (3) A. Ragazzi. Micologia Italiana 1:29, 2009. (4) A. Vannini and R. Valentini. Tree Physiol. 14:129, 1994.

Root Rot and Dieback of Pinus pinea Caused by Phytophthora humicola in Tuscany, Central Italy.

High mortality was noticed in a 10-year-old stand of Pinus pinea in the Alberese area (Grosseto, central Italy, elev. 40 m, 42° 39' 46″ N, 11° 06' 25″ E) in July 2010. Aerial symptoms of trees included chlorosis, crown thinning, stunted growth, bark lesions at the stem base with resinous exudations, and extensive necroses of the underlying xylem tissue. Woody roots of two uprooted trees exhibited bark necroses and a high proportion of fine roots was destroyed. Soil around necrotic roots was baited using apple fruits (cv. Gala). After 1 week of incubation at 24°C, typical firm fruit rot developed and small tissue samples were transferred to clarified V8 agar (V8A) amended with 5 ml/l PARPNH and incubated at 24°C. After 7 days, stellate to rosaceous, finely lobed cottony colonies arose that were transferred to FPM medium and incubated at 24°C. Within 7 days, spherical oogonia with a smooth surface and predominantly paragynous antheridia formed; sporadic amphyginous antheridia could be observed. Colony squares (1 cm2) were then placed in filtered and sterilized pond water. After 48 h, ovoid, obpyriform, or clavate, nonpapillate, persistent sporangia with internal nested and extended proliferation were formed. Fifty oogonia and 30 sporangia were measured. The diameter of the 50 spherical oogonia varied from 33.6 to 44.9 µm (avg. 39 µm); dimensions of the 30 sporangia were 42.6 to 59.8 × 28.9 to 47.8 µm (avg. 52.95 × 38.98 µm; 1:b ratio 1.37). The isolate was identified as Phytophthora humicola W. H. Ko & Ann on the basis of colony type, size and morphology of oogonia and sporangia, average length/width ratio of sporangia, the homothallic formation of oogonia (4), and ITS rDNA sequence information (GenBank Accession No. JQ757060). A BLAST search of the ITS sequence of P. humicola isolate B33 revealed a 99% identity with the Phytophthora ITS Clade 6 species P. humicola and P. inundata (2). This latter species could be ruled out, however, since it is self-sterile, whereas our isolate B33 was self-fertile (3). A strain of P. humicola was deposited in the CBS-KNAW Fungal Biodiversity Centre, strain no. CBS129249. Pathogenicity tests were conducted on 10 one-year-old twigs of Pinus pinea. A bark portion was removed aseptically and a V8A disc (0.5 cm diam.) of P. humicola mycelium was placed on the wound. Control twigs (3) received sterile V8A discs. Inoculated and control twigs were incubated at 20°C in the dark. Clearly noticeable necrotic lesions (avg. length 2.2 × 0.68 cm) were observed after 15 days on inoculated twigs. Control twigs showed no symptoms. Reisolations on selective V8-PARPNH-agar confirmed P. humicola as the causal agent. P. humicola is mainly associated with woody horticultural crops (1, 3), while the other taxa grouped with this species in Clade 6 are mainly found in forest and riparian ecosystems (1). These aquatic Phytophthora species normally have a saprophytic lifestyle, but under favourable environmental circumstances can act as opportunistic pathogens, attacking susceptible trees and causing scattered mortality in forest stands and natural ecosystems (3). To our knowledge, this is the first report of P. humicola from a pine stand. It is supposed that the pathogen reached the stand through infected plant material or infested soil introduced into the stand. References: (1) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) T. Jung et al. Persoonia 26:13, 2011. (4) W. H. Ko and P. J. Ann. Mycologia 77:631, 1985.

Fungal endophytes in Mediterranean oak forests: a lesson from Discula quercina.

Fungal endophytes that colonize forest trees are widespread, but they are less well known than endophytes infecting grasses. The few studies on endophytes in trees mainly concern the tropical areas and the northernmost latitudes, while similar investigations in the Mediterranean region have so far been scarce and incidental. Endophytes are studied mostly in economically important forests suffering from diseases, such as oak forests. One common endophyte that has received some study on oak is the mitosporic Discula quercina. This paper, after first addressing some basic problems on tree endophytes, examines the ecology of D. quercina in Mediterranean oak stands. D. quercina is usually viewed as a symptomless colonizer of healthy Quercus cerris, infecting new leaves early in the growing season, in an unstable equilibrium between transient mutualism/neutralism and latent pathogenesis. It is postulated here that climatic factors can change the endophytic nature of D. quercina, turning it into a weak pathogen or an opportunistic invader of senescing and indeed healthy trees. It is argued more generally that stochastic events can cause the lifestyle of an endophyte to switch from beneficial/neutral to pathogenic, transforming the tree-endophyte interaction, an interaction that depends in part on the matching genomes of the tree and endophyte, and on the environmental context.

First Report of Botryosphaeria dothidea on Sycamore, Red Oak, and English Oak in Northwestern Italy.

The Parco Nord Milano, Italy is a 600-ha green area (45°53'71″N, 9°20'97″E). Since the spring of 2002, extensive and unknown decline was observed on 20-year-old sycamore (Acer pseudoplatanus), red oak (Quercus rubra), and English oak (Q. robur) plantations. The trees showed branch and twig dieback associated with bark cankering along the whole stem and an irregular to wilted crown. A closer inspection of the inner part of the symptomatic trunk and roots revealed a dark bluish tissue discoloration. From the symptomatic trunk and root tissue, a dark gray abundant mycelium was consistently isolated on potato dextrose agar (PDA). The sequenced internal transcribed spacer (ITS) regions of rDNA (GenBank Accession Nos. DQ198265, DQ198266, and DQ198267) showed 99% identity to Botryosphaeria dothidea strain CBS110302 (GenBank Accession No. AY259092). A Fusicoccum sp. with black pycnidia was consistently collected from the cankers. The conidia were hyaline, aseptate, fusiform to narrowly ellipsoidal, with a truncate base (22 × 4.5 µm), referred to as Fusicoccum aesculi (CBS identification), the anamorph of B. dothidea (2). Pathogenicity tests were conducted for all three hosts by stem inoculation on 18-month-old seedlings growing in plastic pots containing a 2:1 turf/sand mixture. Two experiments were conducted using two inoculation techniques. In the first trial, 6-mm-diameter mycelial plugs of B. dothidea were applied to 6-mm-long bark wounds. The same inoculation method was used for application to bark without wounding. Control seedlings were inoculated with sterile agar plugs in a similar fashion as above. Inoculated and control seedlings were kept in a growth chamber and watered once per week. In the second trial, segments of branches 15-cm long were inoculated with 6-mm-diameter B. dothidea plugs (1), with and without any wounding. Control segments of branches were treated with sterile agar plugs in place of fungal mycelium. The branches were incubated at 23°C in moist chambers. For both experiments, the inoculated stem portions were wrapped with Parafilm to prevent desiccation. There were three replicate seedlings per inoculation technique, experiment, and plant species. After 2 months, all seedlings showed bark cankers and pycnidial formation, while the controls were symptomless. An inner dark bluish stem tissue discoloration was observed. The symptoms were more abundant on the segment of branches where the inoculum was applied to the wounded bark. B. dothidea was successfully reisolated, confirming Koch's postulates. To our knowledge, although it is accepted as synonymous of B. berengeriana (2), this is the first report of B. dothidea on sycamore, red oak, and English oak in Italy. The fungus was previously reported in Italy to cause canker on Platanus spp. References: (1) M. E. Sanchez et al. Plant Dis. 87:1515, 2003. (2) B. Slippers et al. Mycologia 96:83, 2004.

Antagonism of the Two-Needle Pine Stem Rust Fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum In Vitro and In Planta.

ABSTRACT Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degrees C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

The in vitro effect of gossypol and its interaction with salts on conidial germination and viability of Fusarium oxysporum sp. vasinfectum isolates.

AIMS: To assess the effect of different concentrations of gossypol (0, 2, 4, 10 and 20 mg l(-1)) in combination with NaCl and Na(2)SO(4) (20 mS cm(-1)) on the conidial germination and viability of Fusarium oxysporum f.sp. vasinfectum (Fov). METHODS AND RESULTS: A multinomial logistic model was developed to estimate the germination probability of Fov. The inhibitory effect was markedly evident at the two highest concentrations of gossypol; it varied among the isolates tested and with time, and it was attenuated by the presence of sodium salts. The inhibition was temporary as the germination probability increased after 8 h. Fluorescent staining revealed that gossypol either killed the conidia or retarded the elongation of the germ tubes. CONCLUSION: Fov showed the ability to overcome gossypol inhibition over time, and the inhibitory effect is reduced under saline conditions. Differential responses among Fov isolates to the presence of gossypol suggest that gossypol tolerance is genetically determined in the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that selecting for high plant gossypol cultivars would have minimal effect on the overall Fov resistance of cotton. A new statistical model was developed to explore the statistical significance of plant-pathogen interactions.

Heterogeneity in intergenic regions of the ribosomal repeat of the pine-blister rusts Cronartium flaccidum and Peridermium pini.

Mixed aeciospore isolates of Cronartium flaccidum and Peridermium pini were obtained from single-tree infections in Britain, Italy and Greece. The 5.8s ribosomal RNA gene and flanking intergenic transcribed spacer regions ITS1 and ITS2 were found to be highly similar between C. flaccidum and P. pini. Within samples heterogeneity was detected at three nucleotide loci in the ITS1 and at four loci in the ITS2 suggesting that several fungal genotypes may occur at a single infection court. The heterogeneity was confirmed by heteroduplex polymorphism analysis of mixed aeciospore products. RFLP of the ribosomal intergenic spacer region 1 (IGS1) amplified from the same templates indicated limited sequence polymorphism in some copies of this repeated locus. Both the sexual and asexual forms of C. flaccidum show evidence of sequence polymorphism in two independent, non-coding regions of the ribosomal gene array. Variation appears to be greater in the sexual form C. flaccidum, than in the monoaecious form P. pini.